Methods for rapidly identifying small organic molecule ligands for binding to biological target molecules

ABSTRACT

The present invention is directed to novel methods for rapidly and unambiguously identifying small organic molecule ligands for binding to biological target molecules. Small organic molecule ligands identified according to the methods of the present invention may find use, for example, as novel therapeutic drug lead compounds, enzyme inhibitors, labeling compounds, diagnostic reagents, affinity reagents for protein purification, and the like. Also presented are novel methods for identifying high affinity binding ligands for a biological target molecule of interest, wherein those methods comprise linking two or more small organic molecule ligands previously identified as being capable of binding to the biological target molecule of interest. Biological target molecules include, for example, polypeptides, nucleic acids, carbohydrates, nucleoproteins, glycoproteins, glycolipids and lipoproteins.

FIELD OF THE INVENTION

The present invention is directed to novel molecular methods useful forquickly and unambiguously identifying small organic molecule ligands forbinding to specific sites on target biological molecules. Small organicmolecule ligands identified according to the methods of the presentinvention find use, for example, as novel therapeutic drug leadcompounds, enzyme inhibitors, labeling compounds, diagnostic reagents,affinity reagents for protein purification, and the like.

BACKGROUND OF THE INVENTION

The primary task in the initial phase of generating novel biologicaleffector molecules is to identify and characterize one or more tightlybinding ligand(s) for a given biological target molecule. In thisregard, many molecular techniques have been developed and are currentlybeing employed for identifying novel ligands that bind to specific siteson biomolecular targets, such as proteins, nucleic acids, carbohydrates,nucleoproteins, glycoproteins and glycolipids. Many of these techniquesexploit the inherent advantages of molecular diversity by employingcombinatorial libraries of potential ligand compounds in an effort tospeed up the identification of functional ligands. For example,combinatorial synthesis of both oligomeric and non-oligomeric librariesof diverse compounds combined with high-throughput screening assays hasalready provided a useful format for the identification of new leadcompounds for binding to chosen molecular targets.

While combinatorial approaches for identifying biological effectormolecules have proven useful in certain applications, these approachesalso have some significant disadvantages. For example, current synthetictechnology is limited in that it allows one to synthesize only arelatively small fraction of the total number of possible librarymembers for any given molecule type. As such, even when appropriatehigh-throughput screening assays are available for screening a library,only a small fraction of the total number of possible members of anymolecule type will be represented in the library and, therefore,screened for the ability to bind to the chosen target. Thus,combinatorial approaches often do not allow one to identify the “best”ligand for a target molecule of interest.

Additionally, even when appropriate screening assays are available, inmany cases techniques which allow identification of the actual librarymember(s) which bind most effectively to the target are not available orprovide ambiguous results, making the actual identification andcharacterization of functional ligand molecules difficult or impossible.

Furthermore, many approaches currently employed to identify novelligands are dependent upon only a single specific chemistry, therebylimiting the usefulness of such approaches to only a narrow range ofapplications. Finally, many of the approaches currently employed areexpensive and extremely time-consuming. Thus, there is a significantinterest in developing new methods which allow rapid, efficient andunambiguous identification of small organic molecule ligands forselected biomolecular targets. It is also desired that such techniquesare adaptable to a variety of different chemistries, thereby beinguseful for a wide range of applications.

Schiff base adduct formation involves the reaction of an availablealdehyde or ketone functionality with a primary amine to form animine-bonded complex. While the Schiff base adduct is relativelyunstable, numerous groups have employed aldehyde or ketone compounds forbonding to primary amine functionalities on proteins of interest for avariety of purposes (see, e.g., Pollack et al., Science 242:1038-1040(1988), Abraham et al., Biochemistry 34:15006-15020 (1995) and Boyiri etal., Biochemistry 34:15021-15036 (1995)). We herein describe noveltechniques useful for rapidly and efficiently identifying organicmolecule ligands that bind to specific sites on biomolecular targets,wherein those techniques are adaptable to a variety of differentchemistries, preferably Schiff base adduct formation between a targetpolypeptide and one or more members of a library of potential organicmolecule ligands. These methods allow one to unambiguously identify andcharacterize the organic molecule ligand that binds most efficiently tothe chosen target. Additionally, the herein described methods are quick,easy to perform and inexpensive as compared to other currently employedmethods.

SUMMARY OF THE INVENTION

Applicants herein describe a molecular approach for rapidly andefficiently identifying small organic molecule ligands that are capableof interacting with and binding to specific sites on biological targetmolecules, wherein ligand compounds identified by the subject methodsmay find use, for example, as new small molecule drug leads, enzymeinhibitors, labeling compounds, diagnostic reagents, affinity reagentsfor protein purification, and the like. The herein described approachesallow one to quickly screen a library of small organic compounds tounambiguously identify those that have affinity for a particular site ona biomolecular target. Those exhibiting affinity for interacting with aparticular site are capable of forming a covalent bond with a chemicallyreactive group at that site, whereby small organic compounds capable ofcovalent bond formation may be readily identified and characterized.Such methods may be performed quickly, easily and inexpensively andprovide for unambiguous results. The small organic molecule ligandsidentified by the methods described herein may themselves be employedfor numerous applications, or may be coupled together in a variety ofdifferent combinations using one or more linker elements to providenovel binding molecules.

With regard to the above, one embodiment of the present invention isdirected to a method for identifying an organic molecule ligand thatbinds to a site of interest on a biological target molecule, wherein themethod comprises:

(a) obtaining a biological target molecule that comprises or has beenmodified to comprise a chemically reactive group, wherein the site ofinterest on the target molecule comprises the chemically reactive group;

(b) combining the target molecule with one or more members of a libraryof organic compounds that are capable of covalently bonding to thechemically reactive group, wherein at least one member of the librarybinds to the site of interest to form a covalent bond with thechemically reactive group to form a target molecule/organic compoundconjugate; and

(c) identifying the organic compound that forms a covalent bond with thechemically reactive group.

In particular embodiments, the biological target molecule is apolypeptide, a nucleic acid, a carbohydrate, a nucleoprotein, aglycopeptide or a glycolipid, preferably a polypeptide, which may be,for example, an enzyme, a hormone, a transcription factor, a receptor, aligand for a receptor, a growth factor, an immunoglobulin, a steroidreceptor, a nuclear protein, a signal transduction component, anallosteric enzyme regulator, and the like. The target molecule maycomprise the chemically reactive group without prior modification of thetarget molecule or may be modified to comprise the chemically reactivegroup, for example, when a compound comprising the chemically reactivegroup is bound to the target molecule.

Other embodiments of the above described methods employ libraries oforganic compounds which comprise aldehydes, ketones, oximes, hydrazones,semicarbazones, carbazides, primary amines, secondary amines, tertiaryamines, N-substituted hydrazines, hydrazides, alcohols, ethers, thiols,thioethers, thioesters, disulfides, carboxylic acids, esters, amides,ureas, carbamates, carbonates, ketals, thioketals, acetals, thioacetals,aryl halides, aryl sulfonates, alkyl halides, alkyl sulfonates, aromaticcompounds, heterocyclic compounds, anilines, alkenes, alkynes, diols,amino alcohols, oxazolidines, oxazolines, thiazolidines, thiazolines,enamines, sulfonamides, epoxides, aziridines, isocyanates, sulfonylchlorides, diazo compounds and/or acid chlorides, preferably aldehydes,ketones, primary amines, secondary amines, alcohols, thioesters,disulfides, carboxylic acids, acetals, anilines, diols, amino alcoholsand/or epoxides, most preferably aldehydes, ketones, primary amines,secondary amines and/or disulfides.

Methods for identifying the organic compound that forms a covalent bondwith the chemically reactive group on the target molecule may optionallyinclude processes that employ mass spectrometry, high performance liquidchromatography and/or fragmenting the target protein/organic compoundconjugate into two or more fragments.

A particularly preferred embodiment of the present invention is directedto a method for identifying an organic molecule ligand that binds to abiological target molecule of interest, wherein the method comprises:

(a) obtaining a biological target molecule that comprises or has beenmodified to comprise a first reactive functionality,

(b) reacting the target molecule with a compound that comprises (1) asecond reactive functionality and (2) a chemically reactive group,wherein the second reactive functionality reacts with the first reactivefunctionality of the target molecule to form a covalent bond, therebyproviding a target molecule comprising the chemically reactive grouplinked to the target molecule through a covalent bond;

(c) combining the target molecule with one or more members of a libraryof organic compounds that are capable of covalently bonding to thechemically reactive group, wherein at least one member of the libraryforms a covalent bond with the chemically reactive group to form atarget molecule/organic compound conjugate; and

(d) identifying the organic compound that forms a covalent bond with thechemically reactive group.

Preferably, the covalent bond formed from reaction of the first andsecond reactive functionalities is a disulfide bond formed between twothiol groups and optionally, subsequent to step (c) and prior to step(d) one may liberate the covalently-bonded organic compounds from theconjugate by treatment with an agent that disrupts the disulfide bond,wherein that agent may comprise, for example, dithiothreitol,dithioerythritol, β-mercaptoethanol, sodium borohydride or a phosphine,such as tris-(2-carboxyethyl)-phosphine (TCEP). In various embodiments,the biological target molecule is as described above, preferably apolypeptide that may be obtained, for example, as a recombinantexpression product or synthetically. The thiol group and thiolfunctionality may be masked or activated

In particularly preferred embodiments, the chemically reactive group isa primary or secondary amine group and the library of organic compoundscomprises aldehydes and/or ketones, preferably aldehydes, or thechemically reactive group is an aldehyde or ketone group, preferably analdehyde, and the library of organic compounds comprises primary and/orsecondary amines, thereby allowing Schiff base adduct formation betweenthe chemically reactive group and members of the library. Subsequent toSchiff base adduct formation but prior to identifying thecovalently-bound organic compound, a reducing agent may optionally beemployed to reduce the imine bond of the Schiff base adduct.

Yet another embodiment of the present invention is directed to a methodfor identifying a ligand that binds to a biological target molecule ofinterest, wherein the method comprises:

(a) identifying a first organic molecule ligand that binds to thebiological target molecule by at least one of the methods describedabove;

(b) identifying a second organic molecule ligand that binds to thebiological target molecule by at least one of the methods describedabove; and

(c) linking the first and second identified organic molecule ligandsthrough a linker element to form a conjugate molecule that binds to thetarget molecule.

Preferably, the biological target molecule is a polypeptide. In certainembodiments, the first and second organic molecule ligands may bind tothe same site on the target molecule or to different sites thereon. Thefirst and second organic molecule ligands may also be from the same orfrom different chemical classes.

Additional embodiments of the present invention will become evident tothe ordinarily skilled artisan upon review of the present specification.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a rapid and efficient method foridentifying small organic molecule ligands that are capable of bindingto selected sites on biological target molecules of interest. Theorganic molecule ligands themselves identified by the subject methodsfind use, for example, as lead compounds for the development of noveltherapeutic drugs, enzyme inhibitors, labeling compounds, diagnosticreagents, affinity reagents for protein purification, and the like, ortwo or more of the identified organic molecule ligands may be coupledtogether through one or more linker elements to provide novelbiomolecule-binding conjugate molecules.

One embodiment of the subject invention is directed to a method foridentifying an organic molecule ligand that binds to a site of intereston a biological target molecule. As an initial step in the hereindescribed method, a biological target molecule is obtained as a targetfor binding to the small organic molecule ligands screened during theprocess. Biological target molecules that find use in the presentinvention include all biological molecules to which a small organicmolecule may bind and preferably include, for example, polypeptides,nucleic acids, including both DNA and RNA, carbohydrates,nucleoproteins, glycoproteins, glycolipids, and the like. The biologicaltarget molecules that find use herein may be obtained in a variety ofways, including but not limited to commercially, synthetically,recombinantly, from purification from a natural source of the biologicaltarget molecule, etc.

In a particularly preferred embodiment, the biological target moleculeis a polypeptide. Polypeptides that find use herein as targets forbinding to organic molecule ligands include virtually any peptide orprotein that comprises two or more amino acids and which possesses or iscapable of being modified to possess a chemically reactive group forbinding to a small organic molecule. Polypeptides of interest findinguse herein may be obtained commercially, recombinantly, synthetically,by purification from a natural source, or otherwise and, for the mostpart are proteins, particularly proteins associated with a specifichuman disease condition, such as cell surface and soluble receptorproteins, such as lymphocyte cell surface receptors, enzymes, such asproteases and thymidylate synthetase, steroid receptors, nuclearproteins, allosteric enzyme inhibitors, clotting factors,serine/threonine kinases and dephosphorylases, threonine kinases anddephosphorylases, bacterial enzymes, fungal enzymes and viral enzymes,signal transduction molecules, transcription factors, proteinsassociated with DNA and/or RNA synthesis or degradation,immunoglobulins, hormones, receptors for various cytokines including,for example, erythropoietin/EPO, granulocyte colony stimulatingreceptor, granulocyte macrophage colony stimulating receptor,thrombopoietin (ITPO), IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IL-11,IL-12, growth hormone, prolactin, human placental lactogen (LPL), CNTF,octostatin, various chemokines and their receptors such as RANTES,MIP1-α, IL-8, various ligands and receptors for tyrosine kinase such asinsulin, insulin-like growth factor 1 (IGF-1), epidermal growth factor(EGF), heregulin-α and heregulin-β, vascular endothelial growth factor(VEGF), placental growth factor (PLGF), tissue growth factors (TGF-α andTGF-β), other hormones and receptors such as bone morphogenic factors,folical stimulating hormone (FSH), and leutinizing hormone (LH), tissuenecrosis factor (TNF), apoptosis factor-1 and -2 (AP-1 and AP-2), mdm2,and proteins and receptors that share 20% or more sequence identity tothese.

The biological target molecule of interest will be chosen such that itpossesses or is modified to possess a chemically reactive group which iscapable of forming a covalent bond with members of a library of smallorganic molecules. For example, many biological target moleculesnaturally possess chemically reactive groups (for example, amine groups,thiol groups, aldehyde groups, ketone groups, alcohol groups and a hostof other chemically reactive groups; see below) to which members of anorganic molecule library may interact and covalently bond. In thisregard, it is noted that polypeptides often have amino acids withchemically reactive side chains (e.g., cysteine, lysine, arginine, andthe like). Additionally, synthetic technology presently allows thesynthesis of biological target molecules using, for example, automatedpeptide or nucleic acid synthesizers, which possess chemically reactivegroups at predetermined sites of interest. As such, a chemicallyreactive group may be synthetically introduced into the biologicaltarget molecule during automated synthesis.

Moreover, techniques well known in the art are available for modifyingbiological target molecules such that they possess a chemically reactivegroup at a site of interest which is capable of forming a covalent bondwith a small organic molecule. In this regard, different biologicalmolecules may be chemically modified (using a variety of commercially orotherwise available chemical reagents) or otherwise coupled, eithercovalently or non-covalently, to a compound that comprises both a groupcapable of linking to a site on the target molecule and a chemicallyreactive group such that the modified biological target molecule nowpossesses an available chemically reactive group at a site of interest.With regard to the latter, techniques for linking a compound comprisinga chemically reactive group to a target biomolecule are well known inthe art and may be routinely employed herein to obtain a modifiedbiological target molecule which comprises a chemically reactive groupat a site of interest.

In one particular embodiment of the present invention, a target moleculecomprises at least a first reactive group which, if the target is apolypeptide, may or may not be associated with a cysteine residue ofthat polypeptide, preferably is associated with a cysteine residue ofthe polypeptide of interest. Preferably, the polypeptide of interestwhen initially obtained or subsequently modified comprises only alimited number of free thiol groups which may potentially serve ascovalent binding sites for a compound comprising a thiol functionality,where in certain embodiments the polypeptide of interest comprises orhas been modified to comprise no more than about 5 free thiol groups,more preferably no more than about 2 free thiol groups, most preferablyno more than one free thiol group, although polypeptides of interesthaving more free thiol groups will also find use. The polypeptide ofinterest may be initially obtained or selected such that it alreadypossesses the desired number of free thiol groups or may be modified topossess the desired number of free thiol groups. With regard to thelatter, “modified to possess” means that the initially selectedpolypeptide of interest has been recombinantly, chemically, or otherwisealtered such that it possesses a different number of free thiol groupsthan when initially obtained.

Those skilled in the art are well aware of various recombinant,chemical, synthetic, or other techniques that can routinely be employedto modify a polypeptide of interest such that it possess a differentnumber of free thiol groups that are available for covalent bonding to asubsequently-added compound comprising a free thiol group. Suchtechniques include, for example, site-directed mutagenesis, where anucleic acid molecule encoding the polypeptide of interest may bealtered such that it encodes a polypeptide with a different number ofcysteine residues (see, e.g., Gloss et al., Biochemistry 31:32-39(1992)). Site-directed (site-specific) mutagenesis allows the productionof variants of an initially obtained polypeptide of interest through theuse of specific oligonucleotide sequences that encode the DNA sequenceof the desired mutation, as well as a sufficient number of adjacentnucleotides, to provide a primer sequence of sufficient size andsequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Typically, a primer of about 20 to 25nucleotides in length is preferred, with about 5 to 10 residues on bothsides of the junction of the sequence being altered. In general, thetechniques of site-directed mutagenesis are well known in the art, asexemplified by publications such as Edelman et al., DNA 2:183 (1983). Aswill be appreciated, the site-directed mutagenesis technique typicallyemploys a phage vector that exists in both a single-stranded anddouble-stranded form. Typical vectors useful in site-directedmutagenesis include vectors such as the M13 phage, for example, asdisclosed by Messing et al., Third Cleveland Symposium on Macromoleculesand Recombinant DNA, A. Walton, ed., Elsevier, Amsterdam (1981). Thisand other phage vectors are commercially available and their use is wellknown to those skilled in the art. A versatile and efficient procedurefor the construction of oligodeoxyribonucleotide directed site-specificmutations in DNA fragments using M13-derived vectors was published byZoller et al., Nucleic Acids Res. 10:6487-6500 (1982)). Also, plasmidvectors that contain a single-stranded phage origin of replication(Veira et al., Meth. Enzymol. 153:3 (1987)) may be employed to obtainsingle-stranded DNA. Alternatively, nucleotide substitutions areintroduced by synthesizing the appropriate DNA fragment in vitro, andamplifying it by PCR procedures known in the art.

The PCR technique may also be used in modifying a polypeptide ofinterest such that it contains a different number of cysteine residuesthan when initially selected. In a specific non-limiting example of PCRmutagenesis, template plasmid DNA encoding the polypeptide of interest(1 μg) is linearized by digestion with a restriction endonuclease thathas a unique recognition site in the plasmid DNA outside of the regionto be amplified. Of this material, 100 ng is added to a PCR mixturecontaining PCR buffer, which contains the four deoxynucleotidetriphosphates and is included in the GENEAMP® kits (obtained fromPerkin-Elmer Cetus, Norwalk, Conn. and Emeryville, Calif.), and 25 pmoleof each oligonucleotide primer, to a final volume of 50 μl. The reactionmixture is overlayered with 35 μl mineral oil. The reaction is denaturedfor 5 minutes at 100° C., placed briefly on ice, and then 1 μl Thermusaquaticus (Taq) DNA polymerase (5 units/μl), purchased from Perkin-ElmerCetus, Norwalk, Conn. and Emeryville, Calif.) is added below the mineraloil layer. The reaction mixture is then inserted into a DNA ThermalCycler (purchased from Perkin-Elmer Cetus) which may be programmed asfollows:

-   -   2 min. 55° C.,    -   30 sec. 72° C., then 19 cycles of the following:    -   30 sec. 94° C.,    -   30 sec. 55° C., and    -   30 sec. 72° C.

At the end of the program, the reaction vial is removed from the thermalcycler and the aqueous phase transferred to a new vial, extracted withphenol/chloroform (50:50 vol), and ethanol precipitated, and the DNA isrecovered by standard procedures. This material is subsequentlysubjected to appropriate treatments for insertion into a vector andexpression of the encoded modified polypeptide.

Other methods for modifying a polypeptide of interest so that itcontains a different number of cysteine residues that when originallyselected include cassette mutagenesis which is based on the techniquedescribed by Wells et al., Gene 34:315 (1985) and phagemid display, forexample, as described in U.S. Pat. No. 4,946,778.

Further details of the foregoing and similar mutagenesis techniques arefound in general textbooks, such as, for example, Sambrook et al.,Molecular Cloning: A Laboratory Manual (New York: Cold Spring HarborLaboratory Press, 1989) and Ausubel et al., Current Protocols inMolecular Biology, Greene Publishing Associates and Wiley-Interscience1991.

In the particular embodiment which employs a biological target moleculecomprising a first reactive functionality, one may directly screen alibrary of organic molecules that are capable of forming a covalent bondwith that first reactive functionality or may covalently bond a compoundto that first reactive functionality which comprises the chemicallyreactive group of interest. With regard to the latter, the targetmolecule comprising the first reactive functionality may be reacted witha compound that comprises (1) a second reactive functionality and (2) achemically reactive group, wherein that compound becomes covalentlybound to the polypeptide of interest. Specifically, the second reactivefunctionality of the compound reacts with the first reactivefunctionality of the target of interest to form a covalent bond, therebyproviding a modified target of interest. Preferably, the first andsecond reactive functionalities are thiol groups, preferably activatedthiol groups, that react to form a covalent bond. The target of interestis “modified” in that it now has covalently bound thereto through acovalent bond the compound that comprises the chemically reactive group.Reaction conditions useful for covalently bonding the compound to thetarget of interest through a covalent bond are known to those skilled inthe art and may employ activating groups such as thiopyridine,thionitrobenzoate, and the like.

The compound that comprises the chemically reactive group may also becovalently bound to the target biomolecule through a covalent bond otherthan a disulfide bond as described above. Those of skill in the art willbe capable of covalently linking a chemically reactive group-containingcompound to a target biomolecule through virtually any type of covalentbond, including the disulfide bond as described above. In this regard,the first and second reactive functionalities may be any chemicallyreactive functionalities that are capable of reacting to form a covalentbond. The reaction between the first and second reactive functionalitiesto form a covalent bond may be the same or different than the reactionbetween the chemically reactive group and library member to form acovalent bond (see below).

For the most part, the compound that bonds to the target biomolecule ofinterest through a covalent, preferably disulfide bond will berelatively small, preferably comprising less than about 20, morepreferably less than about 10, most preferably less than about 5 carbonatoms, although compounds with more carbon atoms may also find useherein. Such compounds will also possess a thiol functionality capableof forming a covalent bond with the free thiol group of the biologicaltarget molecule and may also possess other heteroatoms at certain siteswithin the compound. A particularly preferred compound for use in thisembodiment of the invention is thioethylamine or a derivative thereof,such as 2-amino ethanethiol, which is capable of forming a disulfidebond with the free thiol group of the biological target molecule as wellas providing a chemically reactive amine group for bonding to members ofa library of organic molecules.

The “chemically reactive group” that is either naturally or otherwisepossessed by the biological target molecule or becomes part of thetarget molecule after modification thereof as described above may be anyof a number of different chemically reactive groups and is chosen so asto be compatible with the library of organic molecule compounds thatwill subsequently be screened for bonding at that site. Specifically,the chemically reactive group provides a site at which covalent bondformation between the chemically reactive group and a member of thelibrary of organic compounds may occur. Thus, the chemically reactivegroup will be chosen such that it is capable of forming a covalent bondwith members of the organic molecule library against which it issubsequently screened. In certain specific embodiments, the chemicallyreactive group is either a primary or secondary amine group and thelibrary of organic compounds comprises aldehydes and/or ketones, whereinthe chemically reactive group and the library members are capable offorming covalent bonds. In another specific embodiment, the chemicallyreactive group is either an aldehyde or ketone group and the library oforganic compounds comprises primary and/or secondary amines, wherein thechemically reactive group and the library members are capable of formingcovalent bonds. Using the techniques described above, chemicallyreactive groups may be introduced into specific predetermined sites onthe biological target molecule or may be introduced randomly.

Once a biological target molecule that comprises a chemically reactivegroup of interest is obtained, the biological target molecule is thenused to screen a library of organic compounds to identify those organiccompounds that form a covalent bond with the chemically reactive group.It is expected that those members of the library of organic compoundsthat have the greatest relative affinity for the site on the polypeptidebeing assayed will be those that covalently bond to the chemicallyreactive functionality most abundantly. For example, it has beendemonstrated that allosteric effects in a polypeptide can function todetermine the reactivity of an organic compound for a reactive site onthe polypeptide (see, e.g., Abraham et al., Biochemistry 34:15006-15020(1995)). Thus, it is expected that by screening mixtures of two or moreorganic compounds against a chemically reactive group at a site ofinterest on a target biomolecule, those organic compounds having thehighest non-covalent affinity for the site of interest will be capableof most efficiently forming covalent bonds with the chemically reactivegroup at that site. In this manner, one can determine which librarymembers have the highest relative binding affinity for the site beingtested, wherein that binding affinity is directly related to the abilityof those compounds to form covalent bonds with the chemically reactivegroup at the site of interest.

As described above, the library of organic molecules and the chemicallyreactive group are chosen to be “compatible”, i.e., chosen such thatthey are capable of reacting with one another to form a covalent bond.The library of organic compounds to be screened against the modifiedpolypeptide of interest may be obtained in a variety of ways including,for example, through commercial and non-commercial sources, bysynthesizing such compounds using standard chemical synthesis technologyor combinatorial synthesis technology (see Gallop et al., J. Med. Chem.37:1233-1251 (1994), Gordon et al., J. Med. Chem. 37:1385-1401 (1994),Czarnik and Ellman, Acc. Chem. Res. 29:112-170 (1996), Thompson andEllman, Chem. Rev. 96:555-600 (1996), and Balkenhohl et al., Angew.Chem. Int. Ed. 35:2288-2337 (1996)) and by obtaining such compounds asdegradation products from larger precursor compounds, e.g. knowntherapeutic drugs, large chemical molecules, and the like. Often thecovalent interaction between the chemically reactive group and thelibrary member will be exchangeable, thereby allowing one to identifysmall molecules that bind in the presence of those that do not. Also,exchangeable covalent bonds will be capable of being madenon-exchangeable, thereby “trapping” the small organic ligand that iscovalently bound to the target.

The “organic compounds” employed in the methods of the present inventionwill be, for the most part, small chemical molecules that will generallybe less than about 2000 daltons in size, usually less than about 1500daltons in size, more usually less than about 750 daltons in size,preferably less than about 500 daltons in size, often less than about250 daltons in size and more often less than about 200 daltons in size,although organic molecules larger than 2000 daltons in size will alsofind use herein. Organic molecules that find use may be employed in theherein described method as originally obtained from a commercial ornon-commercial source (for example, a large number of small organicchemical compounds are readily obtainable from commercial suppliers suchas Aldrich Chemical Co., Milwaukee, Wis. and Sigma Chemical Co., St.Louis, Mo.) or may be obtained by chemical synthesis.

Organic molecule compounds that find use in the present inventioninclude, for example, aldehydes, ketones, oximes, such as O-alkyloximes, preferably O-methyl oximes, hydrazones, semicarbazones,carbazides, primary amines, secondary amines, such as N-methylamines,tertiary amines, such as N,N-dimethylamines, N-substituted hydrazines,hydrazides, alcohols, ethers, thiols, thioethers, thioesters,disulfides, carboxylic acids, esters, amides, ureas, carbamates,carbonates, ketals, thioketals, acetals, thioacetals, aryl halides, arylsulfonates, alkyl halides, alkyl sulfonates, aromatic compounds,heterocyclic compounds, anilines, alkenes, alkynes, diols, aminoalcohols, oxazolidines, oxazolines, thiazolidines, thiazolines,enamines, sulfonamides, epoxides, aziridines, isocyanates, sulfonylchlorides, diazo compounds, acid chlorides, and the like, all of whichhave counterpart chemically reactive groups that allow covalent bondformation with the modified polypeptide of interest. In fact, virtuallyany small organic molecule that is capable of covalently bonding to aknown chemically reactive functionality may find use in the presentinvention with the proviso that it is sufficiently soluble and stable inaqueous solutions to be tested for its ability to bind to the biologicaltarget molecule.

Various chemistries may be employed for forming a covalent bond betweenthe chemically reactive group and a member of the organic moleculelibrary including, for example, reductive aminations between aldehydesand ketones and amines (March, Advanced Organic Chemistry, John Wiley &Sons, New York, 4th edition, 1992, pp. 898-900), alternative methods forpreparing amines (March et al., supra, p. 1276), reactions betweenaldehydes and ketones and hydrazine derivatives to give hydrazones andhydrazone derivatives such as semicarbazones (March et al., supra, pp.904-906), amide bond formation (March et al., supra, p. 1275), formationof ureas (March et al., supra, p. 1299), formation of thiocarbamates(March et al., supra, p. 892), formation of carbamates (March et al.,supra, p. 1280), formation of sulfonamides (March et al., supra, p.1296), formation of thioethers (March et al., supra, p. 1297), formationof disulfides (March et al., supra, p. 1284), formation of ethers (Marchet al., supra, p. 1285), formation of esters (March et al., supra, p.1281), additions to epoxides (March et al., supra, p. 368), additions toaziridines (March et al., supra, p. 368), formation of acetals andketals (March et al., supra, p. 1269), formation of carbonates (March etal., supra, p. 392), formation of enamines (March et al., supra, p.1284), metathesis of alkenes (March et al., supra, pp. 1146-1148 andGrubbs et al., Acc. Chem. Res. 28:446-452 (1995)), transitionmetal-catalyzed couplings of aryl halides and sulfonates with alkenesand acetylenes (e.g., Heck reactions) (March et al., supra, pp.717-178), the reaction of aryl halides and sulfonates withorganometallic reagents (March et al., supra, p. 662), such asorganoboron (Miyaura et al., Chem. Rev., 95:2457 (1995)), organotin, andorganozinc reagents, formation of oxazolidines (Ede et al., TetrahedronLetts. 38:7119-7122 (1997)), formation of thiazolidines (Patek et al.,Tetrahedron Letts. 36:2227-2230 (1995)), amines linked through amidinegroups by coupling amines through imidoesters (Davies et al., CanadianJ. Biochem. 50:416-422 (1972)), and the like.

Libraries of organic compounds which find use herein will generallycomprise at least 2 organic compounds, often at least about 25 differentorganic compounds, more often at least about 100 different organiccompounds, usually at least about 300 different organic compounds, moreusually at least about 500 different organic compounds, preferably atleast about 1000 different organic compounds, more preferably at leastabout 2500 different organic compounds and most preferably at leastabout 5000 or more different organic compounds. Populations may beselected or constructed such that each individual molecule of thepopulation may be spatially separated from the other molecules of thepopulation (e.g., each member of the library is a separate microtiterwell) or two or more members of the population may be combined ifmethods for deconvolution are readily available. The methods by whichthe populations of organic compounds are prepared will not be criticalto the invention. Usually, each member of the organic molecule librarywill be of the same chemical class (i.e., all library members arealdehydes, all library members are primary amines, etc.), however,libraries of organic compounds may also contain molecules from two ormore different chemical classes.

Reaction conditions for screening a library of organic compounds againsta chemically reactive group-containing biological target molecule willbe dependent upon the nature of the chemically reactive group and thechemical nature of the chosen library of organic compounds and can bedetermined by the skilled artisan in an empirical manner. For the stepof screening a population of organic molecules to identify those thatbind to a target polypeptide, it will be well within the skill level inthe art to determine the concentration of the organic molecules to beemployed in the binding assay. For the most part, the screening assayswill employ concentrations of organic molecules ranging from about 0.1μM to 50 mM, preferably from about 0.01 to 10 mM, althoughconcentrations outside those ranges may also find use herein.

In a particularly preferred embodiment, the chemically reactive groupthat is linked to the biological target molecule and the library oforganic molecules to be screened against the target molecule are chosensuch that they are capable of reacting to form a Schiff base adduct. ASchiff base adduct is formed from the condensation of aldehydes orketones with primary or secondary amines. Thus, in one embodiment of thepresent invention, the chemically reactive group is a primary orsecondary amine group and the library of organic compounds against whichthe target molecule is screened comprises aldehyde and/or ketonecompounds. In another preferred embodiment, the chemically reactivegroup is either an aldehyde or ketone group and the library of organiccompounds against which the biological target molecule is screenedcomprises primary and/or secondary amines. Once a reversible Schiff baseadduct is formed between the aldehyde or ketone group and the primary orsecondary amine (an interaction that is relatively unstable andreversible), the imine bond created may optionally be reduced (i.e.,made irreversible) by the addition of a reducing agent so as tostabilize the covalently bonded product of the reaction. Such allows oneto identify small organic molecule ligands that bind to the targetprotein in the presence of those that do not. Reducing agents that finduse for such purposes include, for example, sodium cyanoborohydride,sodium triacetoxyborohydride, cyanide, and the like, i.e., agents thatwould not be expected to disrupt any disulfide bonds present on thetarget biomolecule (see, e.g., Geoghegan et al., J. Peptide and ProteinRes. 17(3):345-352 (1981)).

Combining the biological target molecule of interest with one or moremembers of a library of organic compounds will result in the formationof a covalent bond between the chemically reactive group present on thetarget molecule and a member of the organic compound library. Once sucha covalent bond is formed, one may identify the organic compound thatbound in a number of ways. For example, in the case where the chemicallyreactive group was linked to the target biomolecule through a disulfidebond, one may liberate the organic compound from the target molecule bytreatment of the covalently bound complex with an agent that disruptsthe disulfide bond that was formed between the free thiol group of thetarget molecule of interest and the compound that comprises (1) a thiolfunctionality and (2) the chemically reactive group. For the most part,agents capable of disrupting the disulfide bond through which thecovalently bound organic compound is linked to the target molecule ofinterest will be reducing agents such as, for example, dithiothreitol,dithioerythritol, β-mercaptoethanol, phosphines, sodium borohydride, andthe like, preferably thiol-group containing reducing agents.

Once an organic compound that covalently bound to the chemicallyreactive group of the target molecule has been liberated from thecomplex by treatment with an agent that disrupts the disulfide bondthrough which the organic compound is linked, the identity of the actualorganic compound that bound to the target molecule of interest isdetermined by a variety of means. For example, the well known techniqueof mass spectrometry may preferably be employed either alone or incombination with other means for detection for identifying the organiccompound ligand that bound to the target of interest. Techniquesemploying mass spectrometry are well known in the art and have beenemployed for a variety of applications (see, e.g., Fitzgerald andSiuzdak, Chemistry & Biology 3:707-715 (1996), Chu et al., J. Am. Chem.Soc. 118:7827-7835 (1996), Siuzdak, Proc. Natl. Acad. Sci USA91:11290-11297 (1994), Burlingame et al., Anal. Chem. 68:599R-651R(1996), Wu et al., Chemistry & Biology 4:653-657 (1997) and Loo et al.,Am. Reports Med. Chem. 31:319-325 (1996)).

In other embodiments, subsequent to the covalent bonding of the librarymember to the chemically reactive group of the target molecule, thetarget molecule/organic compound conjugate may be directly subjected tomass spectrometry or may be fragmented and the fragments then subjectedto mass spectrometry for identification of the organic compound thatbound to the target molecule. The success of mass spectrometry analysisof the intact target protein/organic compound conjugate or fragmentsthereof will depend upon the nature of the target molecule and can bedetermined on an empirical basis.

In addition to the use of mass spectrometry, one may employ a variety ofother techniques to identify the organic compound that covalently boundto the biological target molecule of interest. For example, one mayemploy various chromatographic techniques such as liquid chromatography,thin layer chromatography, and the like, for separation of thecomponents of the reaction mixture so as to enhance the ability toidentify the covalently bound organic molecule. Such chromatographictechniques may be employed in combination with mass spectrometry orseparate from mass spectrometry. One may optionally couple a labeledprobe (fluorescently, radioactively, or otherwise) to the liberatedorganic compound so as to facilitate its identification using any of theabove techniques. Other techniques that may find use for identifying theorganic compound that bound to the target biomolecule include, forexample, nuclear magnetic resonance (NMR), capillary electrophoresis,X-ray crystallography, and the like, all of which will be well known bythose skilled in the art.

Another embodiment of the present invention is directed to a method foridentifying a ligand that binds to a biological target molecule ofinterest, wherein the method comprises employing the above describedmethods to identify two or more organic molecule ligands that bind tothe target of interest and linking those two or more organic moleculeligands through a linker element to form a conjugate molecule that alsobinds to the target of interest. For the most part, the conjugatemolecule that is comprised of two or more individual organic moleculeligands for the target molecule will bind to the target of interest witha lower dissociation constant than any of the individual components,although such is not a requirement of the invention. The individualorganic molecule components of a conjugate molecule may bind to the samesite or different sites on the target of interest and may be from thesame or different chemical classes. By “same chemical class” is meantthat each component of the conjugate is of the same chemical type, i.e.,each are aldehydes, each are amines, etc.

Linker elements that find use for linking two or more organic moleculeligands to produce a conjugate molecule will be multifunctional,preferably bifunctional, cross-linking molecules that can function tocovalently bond at least two organic molecules together via reactivefunctionalities possessed by those molecules. Linker elements will haveat least two, and preferably only two, reactive functionalities that areavailable for bonding to at least two organic molecules, wherein thosefunctionalities may appear anywhere on the linker, preferably at eachend of the linker and wherein those functionalities may be the same ordifferent depending upon whether the organic molecules to be linked havethe same or different reactive functionalities. Linker elements thatfind use herein may be straight-chain, branched, aromatic, and the like,preferably straight chain, and will generally be at least about 2 atomsin length, more generally more than about 4 atoms in length, and oftenas many as about 12 or more atoms in length. Linker elements willgenerally comprise carbon atoms, either hydrogen saturated orunsaturated, and therefore, may comprise alkanes, alkenes or alkynes,and/or other heteroatoms including nitrogen, sulfur, oxygen, and thelike, which may be unsubstituted or substituted, preferably with alkyl,alkoxyl, hydroxyalkyl or hydroxy groups. Linker elements that find usewill be a varying lengths, thereby providing a means for optimizing thebinding properties of a conjugate ligand compound prepared therefrom.

In yet other embodiments of the present invention, one may obtain atarget molecule/organic molecule conjugate as described above and then“build off” of the first organic compound that covalently bound to thechemically reactive group of the target molecule. For example, the firstorganic compound that covalently bound to the target biomolecule mayitself provide a chemically reactive group to which a second organiccompound may covalently bond. As such, a target biomolecule/organiccompound conjugate may be screened against a library of organic compoundto identify a second organic compound capable of covalently bonding to achemically reactive group on the first organic molecule. This processmay be repeated in an iterative process to obtain progressively higheraffinity organic molecules for binding to the target molecule. Asdescribed above, the first organic compound may itself possess achemically reactive group that provides a site for bonding to a secondorganic molecule or, in the alternative, the first organic molecule maybe modified (either chemically, by binding a compound comprising achemically reactive group thereto, or otherwise) prior to screeningagainst a second library of organic compounds.

Further details of the invention are illustrated in the followingnon-limiting examples.

EXPERIMENTAL

A plasmid containing the thymydilate synthase gene derived from E. coliwill be mutated such that the five normally occurring cysteine residuesare converted to serine residues using site-directed mutagenesis. At thesame time, a single cysteine residue will be engineered into the enzymeactive site. In one case, this could be the normally occurring catalyticcysteine (C146). In another case, this cysteine residue might take theplace of an arginine residue (such as R127) which has been shown not tosignificantly affect the activity of the enzyme when it is mutated(Carreras and Santi, Annu. Rev. Biochem. 64:721-762 (1995)). One canmake any number of different mutant proteins containing a singlecysteine residue in various locations in and around the active site ofthe enzyme. These mutant proteins will be overexpressed and purified aspreviously described (Maley and Maley, J. Biol. Chem. 263:7620-7627(1988)). Generally, the enzyme will be tested for substrate binding and,in the case of the C146 mutant, activity, to ensure that the mutationsdo not significantly perturb the structure of the protein. In all cases,the protein could be subjected to one or more of the following threetreatments.

In the first case, the mutant protein will be reacted with one molarequivalent of a cysteamine/thionitrobenzoic acid mixed disulfide. Thisreagent would be prepared by reacting cysteamine (otherwise known as2-aminoethanethiol) with a thiol activating agent such as5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB) and purifying the productusing the standard techniques of organic chemistry. The protein wouldreact with the reagent to form a new mixed disulfide in which thecysteine group on the protein is attached to the cysteamine moietythrough a disulfide bond. The free primary amine group of the cysteaminewould then be free to react with aldehydes.

In a typical experiment, individual libraries each consisting of a setof ten different aldehydes chosen to be of similar reactivity andstructure will be mixed with the cysteamine-modified protein in aqueousbuffered solution. Initial experiments will dictate the concentration ofaldehydes used; at first, a wide range of different concentrations willbe tested. During this time the aldehyde functionality of individuallibrary members will react with the primary amine group of theprotein-bound-cysteamine to yield an imine. Because this reaction isreversible, equilibrium will favor imine formation with the librarymember that had the highest intrinsic affinity for the active site ofthe protein. After allowing the libraries of aldehydes to react with theprotein for varying lengths of time, the solution will be treated withsodium cyanoborohydride to reduce the imines to secondary amines. Theprotein-cysteamine-compound complex will then be purified away from theunreacted members of the library by using dialysis, chromatography,precipitation, or other methods. Next, the protein will be treated witha disulfide-reducing agent such as dithiothreitol (DTT) ortris-(2-carboxyethyl)-phosphine (TCEP), thereby cleaving the disulfidebond and releasing the captured library member(s) from the protein.These will then be analyzed directly using mass-spectrometry (MS), orthey will first be conjugated to a fluorescent dye (such as fluoresceinby reaction with fluorescein-maleimide) through their thiol moieties andthen analyzed by a combination of chromatography (HPLC or CE) and MS.The later method will allow quantitation of the released librarymembers, and will facilitate analysis if more than a single librarymember bound to the cysteamine-portion of the protein. It should benoted that the initial library can contain more or fewer than tencompounds; the ideal number being determined empirically, and willprobably vary with different combinations of mutants and libraries.

A second methodology will involve reacting thesingle-cysteine-containing mutant protein with athioglycerol/thionitrobenzoic acid mixed disulfide, which will besynthesized analogously to the cysteamine/thionitrobenzoic acid mixeddisulfide described above. Once the thioglycerol is attached to theprotein through a disulfide bond, the modified protein will be treatedwith a 15 mM sodium periodate solution for 15 minutes at roomtemperature (Acharya and Manjula, Biochemistry 26:3524-3530 (1987)) soas to oxidize the glycol portion to an aldehyde. Thisaldehyde-containing protein will then be reacted with librariesconsisting of pools of primary or secondary amines, and the rest of theprocedure would be as described above.

A variation on this second methodology will involve using speciallyconstructed libraries of amines that also contained the glycolfunctionality. After reacting these libraries with the protein andreducing the resulting imines to secondary amines, the proteins will betreated a second time with sodium periodate to oxidize the newlyintroduced glycol to an aldehyde. The protein-compound-aldehyde willthen be reacted with a second amine-containing library and subsequentlyreduced with sodium cyanoborohydride. In principle, this process couldbe repeated several times so as to actually build an organic moleculewithin the active site of the protein. This is similar to the method ofHuc and Lehn (Huc and Lehn, Proc. Natl. Acad. Sci. USA 94:2106-2110(1997)), but with the significant advantage that the molecule is builtselectively into a specified site of interest. Another advantage is thatit is a linear, stepwise process, where we have control over eachindividual step.

Another variation on this second methodology is made possible by thefact that after reduction of the imine a secondary amine is formed, andthis can in principle be reacted with a library of aldehydes. Inpractice, primary amine libraries will be screened against the originalaldehyde-containing protein target, and the amine that binds mosttightly will be identified. This amine alone will then be conjugated tothe aldehyde-containing protein and reduced to form a secondary amine.In other words, a new target protein will be prepared, consisting of theoriginal target protein coupled to the amine selected from the firstlibrary and containing a secondary amine. This new target protein willthen be reacted with a library of aldehydes, and the aldehyde that bindsmost tightly will be identified. There are several advantages to thismethodology. First, as described in the preceding paragraph, it is astepwise approach, where each step can be optimized for speed andaccuracy. Second, two separate libraries are screened so as to maximizethe diversity with a minimum degree of effort. For example, if the aminelibrary and the aldehyde library each contain a mere 1000 members, thenalthough there are one million possible combinations, in practice only1000 of these need to be sampled in order to identify thetightest-binder (i.e., the single tightest-binding amine pre-bound tothe protein and screened against the library of 1000 aldehydes).Finally, this variation requires only simple primary amines andaldehydes or ketones, of which a large number are readily available. Itshould be noted that a similar approach can be used for the first(cysteamine-based) methodology, as that method also has the potential togenerate a secondary amine.

A third methodology will involve reacting the single-cysteine-containingmutant proteins with libraries of disulfides. Because disulfideformation, like imine formation, is reversible, the process should beequilibrium-driven, such that library members that have the highestinherent affinity for the active site will tend to form disulfide bondswith the protein most often. The thiol-disulfide exchange will befurther promoted by adding various concentrations of reduced andoxidized 2-mercaptoethanol so as to fine tune the reactivity. Theprotein will be purified away from the unbound library members andanalyzed as described in the first method.

The foregoing description details specific methods which can be employedto practice the present invention. Having detailed such specificmethods, those skilled in the art will well enough know how to devisealternative reliable methods at arriving at the same information inusing the fruits of the present invention. Thus, however, detailed theforegoing may appear in text, it should not be construed as limiting theoverall scope thereof; rather, the ambit of the present invention is tobe determined only by the lawful construction of the appended claims.All documents cited herein are expressly incorporated by reference.

1. A method for identifying an organic molecule ligand that binds to asite of interest on a biological target molecule, said methodcomprising: (a) obtaining a biological target molecule that comprises orhas been modified to comprise a chemically reactive group, wherein saidsite of interest on said target molecule comprises said chemicallyreactive group; (b) combining said target molecule with one or moremembers of a library of organic compounds that are capable of covalentlybonding to said chemically reactive group, wherein at least one memberof said library binds to said site of interest and forms a covalent bondwith said chemically reactive group to form a target molecule/organiccompound conjugate; and (c) identifying the organic compound that formsa covalent bond with said chemically reactive group.
 2. The methodaccording to claim 1, wherein said biological target molecule isselected from the group consisting of a polypeptide, a nucleic acid, acarbohydrate, a nucleoprotein, a glycopeptide, a glycolipid and alipoprotein.
 3. The method according to claim 2, wherein said biologicaltarget molecule is a polypeptide.
 4. The method according to claim 3,wherein said polypeptide is selected from the group consisting of anenzyme, a hormone, a transcription factor, a receptor, a ligand for areceptor, a growth factor and an immunoglobulin.
 5. The method accordingto claim 1, wherein said biological target molecule comprises saidchemically reactive group without prior modification of said targetmolecule.
 6. The method according to claim 1, wherein said biologicaltarget molecule obtained in step (a) has been modified to comprise saidchemically reactive group.
 7. The method according to claim 6, whereinsaid modification comprises bonding to said target molecule a compoundthat comprises said chemically reactive group.
 8. The method accordingto claim 1, wherein said library of organic compounds comprisesaldehydes, ketones, oximes, hydrazones, semicarbazones, carbazides,primary amines, secondary amines, tertiary amines, N-substitutedhydrazines, hydrazides, alcohols, ethers, thiols, thioethers,thioesters, disulfides, carboxylic acids, esters, amides, ureas,carbamates, carbonates, ketals, thioketals, acetals, thioacetals, arylhalides, aryl sulfonates, alkyl halides, alkyl sulfonates, aromaticcompounds, heterocyclic compounds, anilines, alkenes, alkynes, diols,amino alcohols, oxazolidines, oxazolines, thiazolidines, thiazolines,enamines, sulfonamides, epoxides, aziridines, isocyanates, sulfonylchlorides, diazo compounds and acid chlorides.
 9. The method accordingto claim 1, wherein said library of organic compounds comprises primaryamines, secondary amines, aldehydes or ketones.
 10. The method accordingto claim 1, wherein said chemically reactive group is a primary aminegroup, a secondary amine group, an aldehyde group or a ketone group. 11.The method according to claim 1, wherein step (c) is accomplished by aprocess that employs mass spectrometry.
 12. The method according toclaim 1, wherein step (c) comprises fragmenting said targetmolecule/organic compound conjugate into two or more fragments.
 13. Themethod according to claim 1, wherein subsequent to step (b) and prior tostep (c) said target molecule/organic compound conjugate is combinedwith one or more members of a library of organic molecules that arecapable of covalently bonding to the organic compound previously boundto said target molecule, wherein at least one member of said library oforganic molecules binds to said target molecule/organic compoundconjugate.
 14. A method for identifying an organic molecule ligand thatbinds to a biological target molecule of interest, said methodcomprising: (a) obtaining a biological target molecule that comprises orhas been modified to comprise a first reactive functionality, (b)reacting said target molecule with a compound that comprises (1) asecond reactive functionality and (2) a chemically reactive group,wherein said second reactive functionality reacts with said firstreactive functionality of said target molecule to form a covalent bond,thereby resulting in said chemically reactive group being linked to saidtarget molecule through a covalent bond; (c) combining said targetmolecule with one or more members of a library of organic compounds thatare capable of covalently bonding to said chemically reactive group,wherein at least one member of said library forms a covalent bond withsaid chemically reactive group to form a target molecule/organiccompound conjugate; and (d) identifying the organic compound that formsa covalent bond with said chemically reactive group.
 15. The methodaccording to claim 14, wherein said first and second chemically reactivefunctionalities are activated thiol groups that react to form adisulfide bond.
 16. The method according to claim 15, which furthercomprises subsequent to step (c) and prior to step (d) the step ofliberating the covalently-bonded organic compound from said targetmolecule/organic compound conjugate by treatment with an agent thatdisrupts said disulfide bond.
 17. The method according to claim 16,wherein said agent that disrupts said disulfide bond is dithiothreitol,dithioerythritol, β-mercaptoethanol, sodium borohydride or a phosphine.18. The method according to claim 14, wherein said biological targetmolecule is selected from the group consisting of a polypeptide, anucleic acid, a carbohydrate, a nucleoprotein, a glycopeptide, aglycolipid and a lipoprotein.
 19. The method according to claim 18,wherein said biological target molecule is a polypeptide.
 20. The methodaccording to claim 19, wherein said polypeptide is selected from thegroup consisting of an enzyme, a hormone, a transcription factor, areceptor, a ligand for a receptor, a growth factor and animmunoglobulin.
 21. The method according to claim 19, wherein saidpolypeptide comprises or has been modified to comprise only a singlecysteine residue.
 22. The method according to claim 19, wherein saidpolypeptide is obtained as a recombinant expression product.
 23. Themethod according to claim 19, wherein said polypeptide is syntheticallyderived.
 24. The method according to claim 14, wherein said targetmolecule comprises or has been modified to comprise less than about 2free thiol groups.
 25. The method according to claim 14, wherein saidlibrary of organic compounds comprises aldehydes, ketones, oximes,hydrazones, semicarbazones, carbazides, primary amines, secondaryamines, tertiary amines, N-substituted hydrazines, hydrazides, alcohols,ethers, thiols, thioethers, thioesters, disulfides, carboxylic acids,esters, amides, ureas, carbamates, carbonates, ketals, thioketals,acetals, thioacetals, aryl halides, aryl sulfonates, alkyl halides,alkyl sulfonates, aromatic compounds, heterocyclic compounds, anilines,alkenes, alkynes, diols, amino alcohols, oxazolidines, oxazolines,thiazolidines, thiazolines, enamines, sulfonamides, epoxides,aziridines, isocyanates, sulfonyl chlorides, diazo compounds and acidchlorides.
 26. The method according to claim 14, wherein said chemicallyreactive group is selected from the group consisting of an aldehydegroup and a ketone group and said library of organic compounds comprisesprimary amines and/or secondary amines.
 27. The method according toclaim 14, wherein said chemically reactive group is selected from thegroup consisting of a primary amine group and a secondary amine groupand said library of organic compounds comprises aldehydes and/orketones.
 28. The method according to claim 14, wherein in step (c) onemember of said library of organic compounds reacts with said chemicallyreactive group to form a Schiff base adduct.
 29. The method according toclaim 28, wherein subsequent to step (c) and prior to step (d), saidSchiff base adduct is reduced by addition of a reducing agent.
 30. Themethod according to claim 29, wherein said reducing agent is selectedfrom the group consisting of sodium cyanoborohydride, sodiumtriacetoxyborohydride and cyanide.
 31. The method according to claim 14,wherein said step (d) is accomplished by a process that employs massspectrometry.
 32. A method for identifying a ligand that binds to abiological target molecule of interest, said method comprising: (a)identifying a first organic molecule ligand that binds to saidbiological target molecule by the method of claim 1; (b) identifying asecond organic molecule ligand that binds to said biological targetmolecule by the method of claim 1; and (c) linking said first and secondorganic molecule ligands through a linker element to form a conjugatemolecule that binds to said biological target molecule.
 33. The methodaccording to claim 32, wherein said biological target molecule isselected from the group consisting of a polypeptide, a nucleic acid, acarbohydrate, a nucleoprotein, a glycopeptide, a glycolipid and alipoprotein.
 34. The method according to claim 32, wherein saidbiological target molecule is a polypeptide.
 35. The method according toclaim 34, wherein said first and said second organic molecule ligandsbind to the same site on said polypeptide.
 36. The method according toclaim 34, wherein said first and said second organic molecule ligandsbind to different sites on said polypeptide.
 37. The method according toclaim 32, wherein said first and second organic molecule ligands arefrom the same chemical class.
 38. The method according to claim 32,wherein said first and second organic molecule ligands are fromdifferent chemical classes.
 39. The method according to claim 34,wherein said conjugate molecule binds to said polypeptide with a lowerdissociation constant than either of said first and second organicmolecule ligands.